Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets.
Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by the cytosol fractions of rat platelets. The more polar products are formed via the lipoxygenase pathways in the same way as are 12-HPETE and 12-HETE, since their formation is not inhibited by indomethacin but by eicosa-5,8,11,14-tetraynoic acid (ETYA). The presence of 0.5-1.5mm-reduced glutathione (GSH) in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. l-Cysteine has the same effect as GSH. However, oxidized glutathione (GSSG) and l-cystine are not able to prevent the formation of the more polar products. The results indicate that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE. Thus the presence of GSH in the reaction mixture offers a convenient and precise cell-free assay system for 12-lipoxygenase in rat platelets. Routine assays of 12-lipoxygenase are carried out in the presence of 1mm-GSH in the reaction mixture. The synthesis of 12-HETE by 12-lipoxygenase is linear during the first 4 min of incubation at 37 degrees C, and has a pH optimum of 7.7. The 12-lipoxygenase reaches half-maximal activity at an arachidonate concentration of 20mum. Fractionation of cell homogenates indicates that the cytosol fraction possesses almost all the 12-lipoxygenase activity, whereas the microsomal fraction exhibits little enzyme activity.
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D001091:Arachidonate Lipoxygenases; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D004220:Disulfides / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D066298:In Vitro Techniques; D015289:Leukotrienes; D008084:Lipoxygenase / Q000097:blood*; D016859:Lipoxygenase Inhibitors; D008297:Male; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D001095:Arachidonic Acids; D004220:Disulfides; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D010545:Peroxides; D013438:Sulfhydryl Compounds; D016718:Arachidonic Acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C021201:12-HPETE; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase; D005978:Glutathione